The broad, long-term objective of this proposal is to elucidate the effects of soy foods on metastatic cancers. Metastatic cancer cells in primary tumors, or in the circulation, may establish secondary metastases at distant sites via a plethora of triggers, including dietary and environmental factors. Much work has been conducted on dietary soy in cancer prevention, but not in metastasis of established cancers. Therefore, this proposal addresses a gap in knowledge concerning the role and molecular targets of the major soy isoflavones in metastatic breast cancer. The rationale comes from data generated during the previous SC3 award. We reported that dietary administration of daidzein, or combined genistein, daidzein, and glycitein (5:4:1) in the ratio found in soy foods, increased mammary tumor growth and metastasis in a mouse model. Dietary daidzein or soy isoflavones upregulated gene expression of eukaryotic protein synthesis initiation factors eIF4E and eIF4G, as well as protein expression of pro-cancer molecules sensitive to elevated eIF4E and eIF4G levels. Subsequently, we published that equol, a metabolite of daidzein produced by intestinal bacteria, is a pivotal soy isoflavone that contributes to cancer malignancy by increasing the expression of proteins that govern metastasis. This proposal will build upon the results of the previous award by testing the hypothesis that soy isoflavones regulate cancer progression via regulation of protein synthesis initiation. Human metastatic breast cancer cell lines that represent various breast cancer types: estrogen receptor positive, triple negative, and HER2 type, will be used for the following Specific Aims. Specific Aim 1 will delineate the effects of individual or combined soy isoflavones, genistein, daidzein, equol, and glycitein, on regulation of protein synthesis initiation and cell functions relevant to cancer progression in vitro. This Aim wll also determine the contribution of soy-isoflavone-mediated upregulation of eIF4G and eIF4E to cancer progression by testing the effects of eIF4G or eIF4E knockdown, or rapamycin to inhibit eIF4E. Since equol increased the expression of the central transcription factor c-Myc, a role for c-Myc in upregulation of protein synthesis initiation factors in response to soy isoflavones will also be tested. Specific Aim 2 will validate the role of the daidzein metabolite equol as the key cancer promoting component of soy isoflavones. The effect of equol on metastasis, individually or in combination with genistein and glycitein, will be determined in immunocompromised mice with mammary tumors established from metastatic human breast cancer cells. Mice will also be treated with control or small interfering RNA (siRNA) to eIF4G to investigate a role for eIF4G in equol-mediated tumor growth and metastasis. Extracted tumors and lung metastases from this Aim will also be tested for expression of pro-cancer molecules. This comprehensive experimental strategy is expected to delineate the molecular basis for potential detrimental effects of soy consumption in breast cancer patients and survivors. Therefore, the proposed research is highly relevant to the mission of the National Institute of General Medical Sciences.